Protein glycosylation is an important post-translational modification that regulates a variety of cellular and physiological functions. Glycosylation-related pathologies have been documented in Alzheimer's disease (AD) and are reported to contribute to the etiology and progression of AD. However, the mechanism of disruption and the precise impact of aberrant protein glycosylation in AD are ultimately unknown. The Golgi apparatus, a master regulator of protein glycosylation, is abnormally fragmented in neurons of AD patients, indicating that Golgi fragmentation may be a putative source of abnormal protein glycosylation and neuron dysfunction in AD. The Wang lab has developed a multidisciplinary approach to examine the molecular mechanism and biological significance of Golgi stack formation. We discovered that the Golgi stacking proteins GRASP55 and GRASP65 form trans-oligomers to ?glue? the Golgi cisternae into stacks and provided the first evidence that Golgi stacking regulates protein trafficking to ensure accurate glycosylation and sorting. Notably, we also discovered that accumulation of the toxic, AD-related beta-amyloid (A?) peptide induces GRASP65 phosphorylation, which leads to Golgi unstacking. Significantly, expressing a phosphorylation-deficient mutant of GRASP65 rescues Golgi structure and alters APP trafficking, thereby reducing A? secretion. These results indicate that aberrant regulation of Golgi structural proteins causes Golgi unstacking in AD and exacerbates AD pathology. Recently, we discovered that GRASP55 senses nutrient deprivation through O-GlcNAcylation to promote autophagosome-lysosome fusion. Additionally, we found that GRASP55 O-GlcNAcylation is reduced in an AD cell culture model, which may contribute to autophagy defects in AD. Thus, glycosylation and phosphorylation of Golgi structural proteins potentially link Golgi structural regulation, protein glycosylation, autophagy, and AD pathology. We hypothesize that dysfunctional regulation of Golgi structure and structural proteins contributes to protein glycosylation defects that underlie AD pathogenesis and progression. We propose to apply our expertise on Golgi structure formation, autophagy, and neurodegenerative disease mechanisms to understand the relationship between Golgi fragmentation and abnormal protein glycosylation in AD etiology and progression. In Aim 1, we will determine how Golgi fragmentation contributes to N-glycosylation defects during AD development. Importantly, we will rescue N- glycosylation defects through restoring Golgi structure and function and determine the impact on glycosylation- related AD pathologies. In Aim 2, we will characterize the extent to which Golgi defects impact protein O- GlcNAcylation and autophagy in AD. We will examine the relationship between Golgi fragmentation and GRASP55 O-GlcNAcylation, lysosome enzyme sorting and function, autophagic flux, and APP metabolism. Preventing Golgi-related glycosylation defects may delay AD pathogenesis; thus, our efforts to develop molecular tools that control Golgi structure may suggest rational therapeutic approaches for treatment of AD.